Ca2+-mobilizing hormones and glucagon stimulate hepatic Na+/K+-ATPase. In rat liver, this stimulation occurs fairly rapidly (i.e. less than 30 sec) and is not secondary to increased Na-influx. Evidence from several laboratories including our own has implicated the diacylglycerol/protein kinase C pathway (Ca2+-mobilizing hormones) and cAMP/protein kinase A pathway (glucagon) in the stimulation of the Na+/K+-ATPase by these agents. In view of the potential regulatory role of protein phosphorylation in hormone dependent activation of Na+/K+-ATPase, we propose to test hypothesis that Na+/K+-ATPase is phosphorylated and functionally regulated by two kinases in the regulation of this enzyme two specific aims will be undertaken: The first aim will be to characterize the effect of interventions, which specifically alter protein kinase C and cAMP-dependent protein kinase activity or inhibit protein phosphatases, on the activity of Na+/K+-ATPase as well as the extent of phosphorylation of Na+/K+-ATPase alpha and beta subunits. This will include pharmacologic manipulation of Na+/K+-ATPase activity and phosphorylation with specific activators of cAMP-dependent protein kinase and protein kinase C. The ability specific inhibitors of these two kinases to interrupt hormone dependent activation and phosphorylation of Na+/K+-ATPase will also be studied. Further we will seek to determine whether the protein phosphatase inhibitor, okadiac acid, will prolong or mimic activity and phosphorylation responses to hormones. Freshly isolated rat hepatocytes as well as cell cultures of hepatocytes with down-regulated protein kinase C activities will be utilized for these experiments. In all of the proposed studies, time and concentration dependent changes will be obtained in order to correlate the two responses. Ouabain-sensitive 86Rb+-uptake will be measured to monitor Na+/K+-ATPase enzymatic activity and Na+/K+-ATPase subunit phosphorylation will be evaluated by monitoring the incorporation of 32P into the alpha and beta subunits (i.e. the subunits will be immunoprecipitated from detergent extracts of hepatocytes with [32P] radioequilibrated ATP pools prior to polyacrylamide gel electrophoresis and autoradiography). Preliminary studies have established a two dimensional gel methodology which separates Na+/K+-ATPase subunits and antisera which recognize the subunits. The second aim of these studies is to identify any amino acid residues in Na+/K+-ATPase which are phosphorylated by these kinases and ascertain their location within the primary structure of the alpha- and beta-subunits. For this purpose, immunoprecipitated [32P]-phosphoproteins corresponding to the alpha and beta subunits will be partially hydrolyzed and subjected to phosphoamino acid analysis. I subsequent studies radiolabelled subunits will be subjected to proteolytic digestion prior to electrophoresis, Western blotting and sequence analysis of Na+/K+-ATPase subunits. The long term goals of this project are to elucidate the molecular mechanisms responsible for stimulation of Na+/K+-ATPase by hormones in order to determine their role in the regulation of liver metabolism.